Protein phosphatase 2A holoenzyme is targeted to peroxisomes by piggybacking and positively affects peroxisomal β-oxidation

Abstract

The eukaryotic, highly conserved serine (Ser)/threonine-specific protein phosphatase 2A (PP2A) functions as a heterotrimeric complex composed of a catalytic (C), scaffolding (A), and regulatory (B) subunit. In Arabidopsis (Arabidopsis thaliana), five, three, and 17 genes encode different C, A, and B subunits, respectively. We previously found that a B subunit, B'θ, localized to peroxisomes due to its C-terminal targeting signal Ser-Ser-leucine. This work shows that PP2A C2, C5, andA2 subunits interact and colocalize with B'θ in peroxisomes. C and A subunits lack peroxisomal targeting signals, and their peroxisomal import depends on B'θ and appears to occur by piggybacking transport. B'θ knockout mutants were impaired in peroxisomal β-oxidation as shown by developmental arrest of seedlings germinated without sucrose, accumulation of eicosenoic acid, and resistance to protoauxins indole-butyric acid and 2,4-dichlorophenoxybutyric acid. All of these observations strongly substantiate that a full PP2A complex is present in peroxisomes and positively affects β-oxidation of fatty acids and protoauxins.

Figure 1.

Detection of PP2A catalytic subunit in isolated peroxisomes. A, Catalytic subunit detection in isolated Arabidopsis peroxisomes by western blotting with specific antibodies against methylated (upper) and demethylated (lower) C-terminal end. Two preparations of peroxisomes were used together with Arabidopsis leaf extract (5-week-old plants) as a control. The detected band equals 35 kD. B, Assessment of the purity of isolated peroxisomes using antibodies against cytosolic PEPC and the peroxisomal marker catalase. Perox. prep1, Peroxisomal preparation1; Perox. prep2, peroxisomal preparation2.

Figure 2.

Visualization of B′θ/A2, B′θ/C2, and B′θ/C5 interactions in peroxisomes. Interaction and peroxisomal localization were detected in Arabidopsis mesophyll protoplasts. I, Coexpression of A2::VYNE(R) and B′θ::VYCE(R). II, Coexpression of C2::VYNE and B′θ::VYCE(R). III, Coexpression of C2::VYCE and B′θ::VYNE(R). IV, Coexpression of C5::VYNE and B′θ::VYCE(R) (Leivar et al., 2011). Coexpression of B′θΔSSL::VYCE with C2::VYNE (V) or C5::VYNE (VI) shows cytosolic fluorescence in protoplasts. Peroxisomes were labeled with gMDH-CFP (Fulda et al., 2002). The cyan fluorescence color was converted to red. Scale bars = 5 μM.

Figure 3.

Expression of EYFP-B′θ in peroxisome-like structures in transgenic stable overexpression plants. The F3 progenies of plants transformed by EYFP-B′θ show EYFP fluorescence in punctate structures with variable levels in mesophyll cells (I). The highest expression was seen in roots of 3-d-old seedlings (meristem and elongation zone; II), whereas no such fluorescence was seen in nontransformed plants (III). Scale bars = 5 μm (I) and 200 μm (II and III).

Figure 4.

B′θ expression analyses. A, qRT-PCR shows higher levels of B′θ transcript in senescent leaves, roots, and flowers than in young and mature leaves. Expression levels are given relative to the expression in mature leaves (set to one). Vertical bars represent ses. B, Expression levels of B′θ mRNA (absolute values) in different tissues. The graph was made using Arabidopsis electronic fluorescent pictograph (eFP) browser data (Winter et al., 2007). C, Schematic representation of B′θ encoding gene with position of the T-DNA insertions and TaqMan primers used for analyzing expression. UTR, Untranslated region.

Figure 5.

Evolutionary relationship of PP2A-B′θ homologs. B′θ is conserved in plants, whereas mammals and yeast (Saccharomyces cerevisiae) contain homologs of the B′ family. Arabidopsis B′ family members are highlighted by a red circle. The homologs that have a conserved peroxisome PTS1 are highlighted by a green triangle, whereas the blue triangle marks a change in SSL> to SSS>. The phylogram was generated by MEGA6 (Tamura et al., 2013) using the neighbor-joining method of Saitou and Nei (1987). The tree is drawn with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site.

Figure 6.

Suc dependence assay of b′θ mutants. A, Hypocotyl length of seedlings grown for 6 d in the dark (I and III) or 8 h of light/16 h of dark (II and IV) on one-half-strength Linsmeier and Skoog (LS) medium with or without 1% Suc. The homozygous lines b′θ-1 (I and II) and b′θ-2 (III and IV) were tested. The experiment was repeated three times, and error bars represent sd. B, Representative images showing developmental defects in b′θ-1, b′θ-2, and pex14 (sugar dependence control) seedlings 6 d after germination in short days without Suc (I). Impaired growth was not seen for medium with Suc (II). After 12 d on Suc-free medium, the differences between mutants and the wild type were still clear, but some mutant seedlings started to recover (III and IV). Scale bars = 5 mm.

Figure 7.

Effects of IBA and 2,4-DB on primary root elongation of b′θ-1 seedlings. Plants were grown for 7 d in light on one-half-strength LS medium supplemented with 0.5% Suc and different concentrations of IBA (A), 2,4-DB (B), and IAA (C). The experiment was repeated three times; error bars represent sd.

Figure 8.

Delayed lipid mobilization in b′θ-1 seedlings. Seeds were sown on one-half-strength LS medium without sugar and stratified for 2 d before being transferred to a growth chamber with continuous light at d 0. The relative level of individual fatty acids (A–E) in the wild type Col-0 and b′θ-1 are given as percentages and standardized to the wild type Col-0 levels at d 1. Error bars represent ses (n = 3) of three batches, each with 39 seedlings.

Figure 9.

Brief overview of Arabidopsis PP2A heterotrimer complexity and functions. The PP2A heterotrimer subunits are presented together with functions of PP2A in Arabidopsis (Farkas et al., 2007; Uhrig et al., 2013). Regulation of peroxisomal β-oxidation is a unique function of PP2A that was approved in this study. Several peroxisomal proteins, involved in fatty acid β-oxidation and the glyoxylate cycle, have been shown experimentally to be phosphorylated (Supplemental Table S1), as highlighted in red for the type of phosphorylated amino acid (S, Ser; T, Thr; and Y, Tyr). As highlighted by the red arrows, β-oxidation steps including LACS5 and KAT1 were found to be potential substrates for a peroxisomal PP2A heterotrimer based on the differences between phosphoproteomes of the b’θ mutant and the wild type (this work). For more details, see Table I. The protein abbreviations are as follows: ACX, acyl-CoA oxidase; AIM, enoyl-CoA hydratase; CSY, citrate synthase; ICL, isocitrate lyase; MDH, malate dehydrogenase; MFP, multifunctional protein; and MLS, malate synthase. In glyoxysomes, the acetyl-CoA produced by fatty acid β-oxidation is used as a substrate for the glyoxylate cycle, where succinate is produced and transferred to mitochondria to be metabolized for energy production. Moreover, the peroxisomal ATP-binding cassette transporter (PXA1) and the PEX5, PEX13, and PEX14 were found to be phosphorylated (Supplemental Table S1). The pathway of fatty acid β-oxidation and glyoxylate cycle is modified from the online database (Karp et al., 2005).